Bladder cancer is the sixth most common cancer in the world. The symptoms include microscopic or macroscopic hematuria, painful urination and polyuria; however, none of these symptoms is specific for the disease. The gold standard for diagnosing bladder cancer is cystoscopy and subsequent transurethral resection of the bladder tumour (TURBT). The sensitivity of cystoscopy for non-muscle invasive bladder cancer (NMIBC; stage Ta, Tl and Tis) is around 80% with white-light cystoscopy and >95% with fluorescence (hexaminolevulinate)-guided cystoscopy.
The majority of bladder tumour patients (70-80%) are diagnosed with NMIBC, which has a relatively good prognosis. However, the recurrence rate for these tumours is very high, with around 70 of the patients experiencing relapses, and up to 25% of these recurrences will progress to muscle invasive cancers (MIBC; stage T2-4) with a poor prognosis. The high recurrence rate and the risk of progression require life-long surveillance with periodic cystoscopy, making bladder cancer the most expensive cancer to treat (Avritscher et al., 2006). As less than 10% of all patients presenting with microscopic or visible hematuria will be diagnosed with bladder cancer, the number of cystoscopies performed to rule out bladder cancer is high and places a considerable burden on the healthcare system. Moreover, as cystoscopy is an invasive method that causes considerable discomfort to the patients, there is an unmet need for noninvasive techniques for reliable and cost-effective diagnosis and surveillance of bladder cancer.
Voided urine from bladder tumour patients may contain exfoliated tumour cells that can be identified by cytology. Urine cytology has been used for decades and is still the most common noninvasive technique for detection of bladder tumours. However, it has a low sensitivity for detection of NMIBC (10-20%) Several alternative non-invasive tests have been developed, including some that have been approved by the U.S. Food and Drug Administration (FDA): Bladder tumour antigen assay, NMP22, ImmunoCyt and Urovysion. To date, none of these tests has achieved widespread use in clinical practice due to low specificity (Liou, L. S. (2006). Urothelial cancer biomarkers for detection and surveillance. Urology 67, 25-33; Tetu, B. (2009). Diagnosis of urothelial carcinoma from urine. Mod. Pathol. 22 Suppl 2, S53-S59; Wadhwa, N., Jatawa, S. K., and Tiwari, A. (7017) Non-invasive urine based tests for the detection of bladder cancer. J. Clin. Pathol. 65. 970-975.).
Bladder tumour cells contain a large number of genome alterations, including gross chromosomal aberrations, amplifications, deletions, single nucleotide substitutions and aberrant DNA methylation. Only a minority of the changes found in individual tumours may be required for initiating and maintaining neoplastic growth (“drivers”), with the remainder being “passenger” events that have no or little effect on the malignant phenotype. Both driver and passenger events may have a potential as biomarkers for bladder cancer, provided that they are cancer specific (i.e., not found in normal tissues or present at a different level of expression) and recurrent (i.e., occur in independently arising tumours at appreciable frequencies).
The most frequently mutated genes in bladder cancer include the proto-oncogenes FGFR3, RAS, and PIK3CA, and the tumour suppressor gene TP53. Mutations in FGFR3 are common in NMIBC, with reported frequencies of >60%, whereas TP53 mutations are predominantly found in MIBC. In addition, hundreds of genes have been shown to be differentially methylated between bladder tumours and normal bladder epithelium.
Studies over the last decade have shown that it is possible to detect bladder tumour-specific genome alterations in DNA isolated from urine sediments. The sensitivity and specificity of DNA-based bladder tumour detection vary considerably among studies, depending on the patient population, the choice of DNA biomarkers and the methods employed for detecting these biomarkers. Some studies have reported diagnostic sensitivities close to or above 90% and specificities close to 100% (Dulaimi et al (2004). Detection of bladder cancer in urine by a tumor suppressor gene hypermethylation panel. Clin. Cancer Res. 10, 1887-1893; Costa er al (2010). Three epigenetic biomarkers, GDF15, TMEFF2, and VIM, accurately predict bladder cancer from DNA-based analyses of urine samples. Clin. Cancer Res. 16, 5842-5851; Hoque et al (2006). Quantitation of promoter methylation of multiple genes in urine DNA and bladder cancer detection. J. Natl. Cancer Inst. 98, 996-1004; Reinert et al (2011). Comprehensive genome methylation analysis in bladder cancer: identification and validation of novel methylated genes and application of these as urinary tumor markers. Clin. Cancer Res. 17, 5582-5592). A recent study has suggested that analysis of DNA biomarkers in urine can also be used to monitor recurrence and reduce the number of cystoscopies in low-risk patients with no concomitant tumour (Reinert et al (2012). Diagnosis of bladder cancer recurrence based on urinary levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 hypermethylation. PLoS. One. 7, e46297). With the advent of improved methods for detection of low-abundant, tumour-specific DNA, including third-generation PCR (digital PCR) and next-generation sequencing, the potential of urine-based detection of bladder tumours has increased dramatically.
One of the main challenges when using urinary DNA markers for diagnosis and surveillance of bladder cancer is to obtain a sufficient number of cells for downstream analysis. In some studies, up to 35% of the samples have been excluded from analysis due to insufficient amounts of DNA (Reinert et al., 2012). The number of tumour cells exfoliated into the urine shows a high inter- and intra-individual variability. In general, the number of cells released correlates with tumour size and stage, such that small early-stage tumours will release fewer cells than MIBC. This limits the usefulness of urinary DNA markers in the non-invasive detection and monitoring of disease and disease progression.